Ulrik Boas and Peter M. H. Heegaard- Dendrimers in drug research

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Dendrimers in drug research

Ulrik Boas* and Peter M. H. Heegaard

Department of Immunology and Biochemistry, Danish Veterinary Institute, Blowsvej 27,

Copenhagen, DK-1790, Denmark. E-mail: [email protected]

Received 20th August 2003

First published as an Advance Article on the web 5th December 2003

Dendrimers are versatile, derivatisable, well-defined, compartmentalised chemical polymers with sizes andphysicochemical properties resembling those of biomolecules e.g. proteins. The present critical review (citing 158references) briefly describes dendrimer design, nomenclature and divergent/convergent dendrimer synthesis. Thecharacteristic physicochemical features of dendrimers are highlighted, showing the effect of solvent pH and polarity ontheir spatial structure. The use of dendrimers in biological systems are reviewed, with emphasis on the biocompatibilityof dendrimers, such as in vitro and in vivo cytotoxicity, as well as biopermeability, biostability and immunogenicity. Thereview deals with numerous applications of dendrimers as tools for efficient multivalent presentation of biologicalligands in biospecific recognition, inhibition and targeting.

Dendrimers may be used as drugs for antibacterial and antiviral treatment and have found use as antitumor agents.The review highlights the use of dendrimers as drug or gene delivery devices in e.g. anticancer therapy, and the designof different hostguest binding motifs directed towards medical applications is described.

Other specific examples are the use of dendrimers as glycocarriers for the controlled multimeric presentation ofbiologically relevant carbohydrate moieties which are useful for targeting modified tissue in malignant diseases fordiagnostic and therapeutic purposes. Finally, the use of specific types of dendrimers as scaffolds for presenting vaccineantigens, especially peptides, for use in vaccines is presented.

1 Introduction Dendrimer types and history

Dendritic structures are found widely in nature. These hyper-branched structures have the advantage that they display a desiredmotif in a multivalent fashion, in order to give synergistic

enhancement of a particular function. Recently, a scientific reporton the dry adhesion of Geckos feet to surfaces interestinglyrevealed that the Gecko foot is built up by a dendritic network offoot hairs, ending in millions of foot-hairs which create anextraordinary strong adhesion due to multiple van der Waals forcesbetween each foot hair and the surface.1 In synthetic organicchemistry, dendritic structures emerged in a new class of polymers

named cascade molecules, first reported by Vgtle and his group.2Later on, development of these molecular designs together withadvanced synthetic techniques gave rise to larger dendriticstructures,35 and this class of molecules was renamed dendrimers.The word dendrimer arises from the Greek dendron, meaning tree

or branch, and meros meaning part.6

Other names for den-drimers are arboroles or cascade polymers. Dendrimers are,despite their large molecular size, structurally well-defined, with alow polydispersity in comparison with traditional polymers. On amolecular level the dendritic branching results in semi-globular toglobular structures, mostly with a high density of functionalities onthe surface together with a small molecular volume. The higher

Ulrik Boas received his MSc Degree in organic chemistry from the

University of Copenhagen in 1996. After a period as a research

scientist in the biotech industry he returned to academe and

received a PhD degree in organic and macromolecular chemistry

in 2002. He is currently working as a research scientist at the

Danish Veterinary Institute. His fields of interest are methoddevelopment in organic synthesis and the use of dendrimers in

immunology and protein chemistry.

Peter Heegaard received his MSc degree (biochemistry) from the

University of Copenhagen in 1985 and a PhD in protein chemistry

from the same university in 1992. He has since worked as an

immunologist/protein chemist in infection biology projects at the

University of Copenhagen and, presently, at the Danish Veterinary

Institute. His is currently occupied with prion diagnostics, acute phase responses in various infection models and peptide and

dendrimer immunochemistry. He has published around 50 pa-

pers.

Ulrik Boas (left) and Peter Heegaard (right)

T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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generation dendrimers occupy a smaller hydrodynamic volume

compared to the corresponding linear polymers, due to their

globular structure. However, in comparison with globular proteins,

the dendrimers have a bigger hydrodynamic volume.

The dendritic structure is characterised by layers between eachfocal point (or cascade) called generations (shown as circles on Fig.

1). The exact numbering of generations has been the subject of

some confusion (see e.g. reference 7). In this review, the dendrimer

generation is defined as the number of focal points (cascade points)when going from the core to the surface, a generation 5 (G5)

dendrimer thus has 5 cascade points between the core and the

surface.

The core is sometimes denoted generation zero (G0), as nocascade points are present. For a polypropylene imine (PPI)

dendrimer, the core is 1,4-diaminobutane which has no cascade

points, for a polyamido amine (PAMAM) Starburst dendrimerthe core is ammonia etc. (hydrogen substituents are not considered

a focal point). In PAMAM dendrimers the intermediate compounds

having carboxylate surface groups are denoted half-generation

dendrimers, that is dendrimers ofe.g. G1.5 or G2.5.

The dendrimer design can be based on a large variety of linkages,

such as polyamines (PPI dendrimers),2 a mix of polyamides and

amines (PAMAM dendrimers)4 or built up by more hydrophobic

poly(aryl ether) subunits.8 More recent examples are dendrimer

designs based on carbohydrate9 or calixarene core structures,10 or

containing third period elements like silicon or phosphorus,11justto give a few examples, Fig. 2. Due to the vast number of dendrimer

designs and synthetic approaches used to create these impressive

structures, the present review will have as the main focus, the

biomedical uses of the commercially available PAMAM and PPI

dendrimers and their interaction with biological systems.

1.1 A brief depiction and nomenclature of dendrimers

The full picture of e.g. hostguest interactions involving den-drimers can become severely crowded and confusing. When

considering surface-modified dendrimers and binding of guest

molecules either to the surface or to the outer shell, a briefer

depiction of the dendrimer can be applied. Instead of drawing the

complete dendrimer structure, the inner shells are depicted as a

blackball (see Fig. 3); an italic number beneath the ball is thenumber of functional groups on the depicted surface. In this way the

hostguest interactions become clearer. For a G5-PPI dendrimerthis number will be 64, when only the terminal amino groups are

depicted. The number of outer shell functionalities (pincers) in a

G5-PPI dendrimer will be 32.

Fig. 1 Common commercially available dendrimers. Top left: Polypropylene imine dendrimer (G5). Top right: Polyamido amine dendrimer (G3). Bottom:Polyamido amine (Starburst) dendrimer (G5). Each generation is marked with a circle.

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1.2 Chemical synthesis of dendrimers

Dendritic structures are chemically synthesised by two different

approaches, either divergent or convergent, Fig. 4. In the divergent

approach the dendrimer is synthesised from the core as the starting

point and built up generation by generation. However, the high

number of reactions which have to be performed on a single

molecule (with a large number of equivalent reaction sites),demands very effective transformations (99+% yield) to avoid

defects. Even for very efficient transformations per generation the

yield of perfect G5-PPI dendrimer will only be approximately25% by the divergent method.22,23 The alternative convergent

approach developed by Hawker and Frecht13,24 starts from thesurface and ends up at the core, where the dendrimer segments

(dendrons) are coupled together. In the convergent approach, only

a small number of reactive sites are functionalised in each step,

giving a small number of possible side-reactions (or missingreactions) per step. Each synthesised generation of dendrimer can

therefore be purified, although purification in the higher-generation

dendrons becomes more cumbersome, because of increasing

similarity between reactants and formed product. However, with

proper purification after each step, dendrimers without defects can

be obtained by the convergent approach, Fig. 4.

By the convergent method it is also possible to create intriguing

asymmetric dendrimeric structures for example by joining two

different dendronic segments together in a controlled fashion.25

1.3 The dendrimer structure and the intrinsic propertiesof its compartments

As the dendritic structure grows, several compartments arise. The

dendrimer structure can be divided into three parts:

The multivalent surface, with a high number of potentialreactive sites.

The outer shell just beneath the surface having a well-definedmicroenvironment protected from the outside by the dendrimer

surface.

The core, which in higher generation dendrimers is protectedfrom the surroundings, creating a microenvironment surrounded by

the dendritic branches.26

The interior is thus well-suited for encapsulation of guest

molecules. The three parts of the dendrimer can be tailored

specifically for the desired purposes, e.g. as dendritic sensors, drug

vehicles or drugs. The multivalent surfaces on a higher-generation

dendrimer can contain a very high number of functional groups.This makes the dendritic surfaces and outer shell well-suited to

hostguest interactions where the close proximity of a large numberof species is important.

1.4 Physicochemical properties of dendrimers

Already early in the history of dendrimers it was suggested that the

3-dimensional nanosized structure of the higher generation den-

drimers would make this class of synthetic molecules suitable as

mimics of proteins.27 It must be kept in mind, however, that in

contrast to proteins which consist of folded, linear polypeptide

chains, the branched architecture of the dendrimer interior is to a

large extent formed by covalent bonds, resulting in a somewhat less

flexible structure. In addition, the dendrimer is on average lesscompact than a protein, i.e. interior is not packed as efficiently as in

typical proteins, and the dendrimer contains a substantially higher

number of surface functional groups than proteins of comparable

molecular weight, Table 1).

Fig. 2 Various designs of dendrimers. Top, left: Unimolecular micelle.12 Top, right: Poly aryl ether dendrimer.13 Bottom, left: Polylysine.14 Bottom, middle:

Carbohydrate dendrimer.9 Bottom, right: Silicon based dendrimer.15

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Molecular dynamic studies carried out by several research

groups on dendrimers show that the dendrimers, similar to proteins,

can adapt native (e.g. more tight) or denaturated (e.g. extended)

conformations dependent on the polarity, ionic strength and pH ofthe solvent. Amino-terminated PPI and PAMAM dendrimers (that

is dendrimers having primary amines as surface groups) exhibit

extended conformations upon lowering of pH because electrostatic

repulsion between the protonated tertiary amines in the interior as

well as between the primary amines at the dendrimer surface, forces

the dendrimer branches apart28 (Fig. 5). At pH > 9 back-folding

occurs as a consequence of hydrogen bonding between the interior

protonated tertiary amines and the primary surface amines,resulting in a denser core.29 The pH-related conformational

changes are dependent on the nature of the charged group at the

dendrimer surface. For PPI dendrimers having surface carboxylic

groups small angle neutron scattering (SANS) and NMR

Fig. 3 Black ball depiction of, left: G5-PPI dendrimer; right: G2.5-PAMAM dendrimer.

Fig. 4 Dendrimer synthesis (schematically depicted). Top: Divergent strategy. Bottom: Convergent strategy

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measurements of the diffusion coefficients in aqueous buffer, show

that these dendrimers have the most extended conformations at pH

4 and pH 11, Fig. 5. This may be due to electrostatic repulsionbetween the protonated cationic inner tertiary amines at low pH,

and electrostatic repulsion between the negatively charged deproto-

nated carboxylates at the dendrimer surface at high pH forcing the

dendritic branches apart.

At pH 6 the carboxy-terminated PPI dendrimer has no net charge,

resulting in a tighter conformation controlled by intramolecular

hydrogen bonding.30 Molecular density measurements at this pH

show a homogeneous molecular density over the whole dendrimer,

indicating a substantial degree of back-folding i.e. hydrogen

bonding between terminal groups and groups in the core region,

Fig. 6.

The polarity of the solvent greatly influences the 3-dimensional

structure of dendrimers, and has been subject for conflicting views

and discussions. Initial theoretical studies by de Gennes and Herveton unmodified PAMAM (Starburst) dendrimers using a self-consistent mean-field model, concluded that in good solvents (that

is solvents with a high ability to solvate the dendritic structure), the

dendrimers had the highest molecular density at the periphery,

leading to dense packing of the surface groups upon increasing

generation.31 Calculations by Lescanet and Muthukumar proposed

a uniform molecular density throughout the dendrimer, indicative

of a pronounced degree of backfolding.32 Later, calculations by

Murat and Crest concluded, contrary to de Gennes, that the highest

molecular density in PAMAM dendrimers was located near the

core, independent of the solvent conditions.33

Recent NMR studies performed on PPI dendrimers indicate that

an apolar solvent such as benzene will favour polar intramolecular

interactions (e.g. hydrogen bonding) resulting in back-folding ofthe dendrimer arms into the dendrimer interior, whereas the

increased acidity of chloroform, will increase solvation of the

dendrimeric structure via hydrogen bond donation to the interior

tertiary amines resulting in a more extended conformation of the

dendrimer.34 Both theoretical as well as experimental studies on

amino functionalised PPI and PAMAM dendrimers reflect the

tendency of an apolar solvent (poor solvent) to induce a higher

molecular density in the core region due to back-folding (intra-

molecular polar interactions), and lower molecular density at the

surface. In polar solvents the dendrimer arms are solvated and the

molecular density at the dendrimer surface is increased.35

NMR and SANS studies performed by De Schryvers group onaryl ether dendrimers with a rubicene core, show that these

dendrimers have extended conformations in a p-interacting solventsuch as toluene, whereas a polar solvent such as acetonitrile induces

a conformational collapse, probably due to strong intramolecularp-interactions.36 In surface modified PPI dendrimers capable of

hydrogen bonding between surface functionalities (end groups), the

intramolecular hydrogen bonding tends to be enhanced upon

increasing generation of the dendrimeric system, as a consequence

of closer vicinity of the surface functionalities in the highergeneration dendrimers.3739 Furthermore, experimental studies

show that the hydrogen bonding end groups are located at the

periphery of the dendrimer, supporting de Gennes dense shellpacking model.40

A microenvironment can arise in the dendrimer core as a

consequence of limited diffusion of solvent molecules into the

dendrimer. As an example, dendrimers dissolved in polar solvents

such as aqueous media can have a very apolar interior (unim-

olecular micelle) allowing organic molecules to be encapsulated

and carried in aqueous media. As we shall see, this property of

dendrimers makes this class of molecules very well-suited as

carriers of various bioactive substances.

2 Properties of dendrimers in biological systems

2.1 The significance of multivalency in biologicalinteractions

Multivalent interactions can be found throughout nature, ranging

from the divalent binding of antibodies and many biological

receptors to the multimillion-valent interactions of a Geckos foot-hair.1 Multivalency has been shown to lead to a strongly increased

activity compared to the corresponding monomeric interaction.

This synergistic enhancement of a certain activity e.g. catalytic

activity or binding affinity from a monomeric to a multimeric

system, is generally referred to as the cluster- or dendritic

effect.9,4143 The dendritic effect is attributed to a co-operativeeffect in a multivalent system leading to a larger increase in activity

than expected from the valency of the system (i.e. additive

increase). It is thus important to differentiate between different

phenomena:

with a higher number of binding entities per molecule in adendrimer substance there is a simple increase in the mole to mole

efficiency of binding (one mole of ligand corresponds to several

moles of binding entities), i.e. an additive effect;

a dendritic effect (or cluster-effect) comes into play when thesimultaneous attachment to n binding entities in the same ligand

molecule leads to an synergistic increase in affinity with a

maximum binding affinity of (single ligand affinity)n. The cluster

effect has especially been observed for carbohydrateprotein

receptors in natural systems with the glycoside cluster effect as aclassic example.44

It is important to realise that multivalency can also increase the

specificity of a given interaction,41 essentially by increasing both

the (high) affinity for the specific ligand and the (low) affinity for

Table 1 Comparison between dendrimers and biological entities. Selected physicochemical parameters

Type of moleculeMolecularweight

pI/surfacecharge Diameter

Number and typeof surface functional groupsa

G3-PAMAM (Starburstb) 2411 /+ 2.2 nm16 12 primary aminesG6-PAMAMc 28 788 11/+17 6.5 nm18 128 primary aminesG6-PAMAM-OH 28 913 9/017 128 hydroxylsMedium sized protein (ovalbumin) 43 000 5/+ and 2 5 nm21 20 primary amines

10 phenol groups4 thiols, 7 imidazoles19

Large protein (Keyhole LimpetHemocyanin)

~ 5 000 000 /+ and 2 approx. 2000primary amines, 700 thiols, 1900 phenols20

Virus21 ~ 40000 000 50200 nm Prokaryotic bacteria21 mainly negative 12 mm (30 nm cell membrane

and cell wall)

Eukaryotic cell21 mainly negative 20 mm (9 nm cell membrane) a protein functional groups not necessarily surface localised b core group is trifunctional, branches are made up of tris(aminoethyl)amine, methyl acrylate andethylenediamine building blocks; Starburst is a Trademark of Dendritech Inc., Midland, MI, US. c Core group is tetrafunctional, branches are made up ofmethyl acrylate and ethylenediamine building blocks.

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the unspecific ligand geometrically, leading to a much higher ratio

of the specific affinity compared to the unspecific affinity.

Biological systems are replete with examples of multivalent

interactions45 and this can be rationalised by the fact that

multivalent interactions provide:

tight binding from rather low-affinity binding of singleligands;

a possibility of utilising low-affinity ligands in a newarrangement to cope with an evolutionary new binding partner;

more efficient cell-cell interactions mediated by multipleinteractions.

Factors that play a role in the binding of dendrimeric multivalent

ligands include obviously the geometry of the multimerically

presented ligands and the flexibility of their attachment to e.g. adendrimer, Fig. 7.46

Dendrimers are perfectly suited to supply multivalency of

synthetic or semisynthetic, biologically interesting entities in a

spatially well-defined manner.

2.2 Biocompatibility of dendrimers

In order to apply dendrimers as tools for drug design or as drug

delivery devices in vivo, they have to fulfil several biological

demands of crucial importance. The dendrimers should be:

non-toxic; non-immunogenic (if not required e.g. for vaccines); able to cross biobarriers such as, e.g. intestine, blood-tissue

barriers, cell membranes etc.;

able to stay in circulation for the time needed to have a clinicaleffect;

able to target to specific structures.

2.2.1 In vitro toxicity. Dendrimers with cationic surfacegroups: Not only dendrimers but cationic macromolecules in

general cause destabilisation of the cell membrane and result in cell

lysis.47 The exact mechanism of cytotoxicity caused by these

polycationic structures has not yet been fully revealed.

Fig. 5 Top row: Three dimensional depiction of conformational change of an amino-terminated PAMAM dendrimer at increasing pH (reprinted with kindpermission from reference 28, copyright (2002) American Chemical Society). Middle row: Two-dimensional depiction of the conformational change of anamino-terminated PAMAM dendrimer upon increasing pH. Bottom row: Two-dimensional depiction of the conformational change of a carboxy-terminatedPPI dendrimer at increasing pH.30

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Initially, comparative toxicity studies on different cell-lines

concluded that amino-terminated PAMAM (Starburst) den-drimers had lower cytotoxicity than the lysine based dendrimer,

polylysine 115 with LD50 = 25 mg mL21 and LD50 > 300 mgmL21 for polylysine 115 and G6-PAMAM, respectively.48 How-

ever, later in vitro cytotoxicity by IC50 measurements (the

concentration where 50% inhibition of mitochondrial dehy-

drogenase activity is measured) on amino-terminated PAMAM

dendrimers showed a significant cytotoxicity of this class ofcompounds on human intestinal adenocarcinoma Caco-2 cells,

albeit, with some variation in IC50 values.49,50 In addition, the

cytotoxicity has shown to be generation dependent for amino-

terminated PAMAM dendrimers, with the higher generation

dendrimers being the most cytotoxic.16,49,51 This is in accordance

with the general finding that increasing molecular size of polymers

may result in increased cytotoxicity.51 Also, haematotoxicity

studies using amino-terminated PAMAM dendrimers conclude that

the dendrimer has haemolytic effect on a solution of rat blood cells

which increases with increasing dendrimer generation.52 Myotox-

icity studies on rodent muscles isolated from male Sprague Dawley

rats showed that the amino-terminated G4-PAMAM dendrimer was

more myotoxic than cationic liposomes and proteins.53 Other

studies on neuroblastoma cells in culture incubating with up to 7.4mg mL21 of PEI, PPI and PAMAM dendrimers for 1 week showedcytotoxicity of the amino-terminated PAMAM and PPI den-

drimers.7

Recent studies have shown that amino-terminated PAMAM

dendrimers, with their globular and less flexible structures, have

lower toxicity than more flexible amino functionalised linear

polymers. This can be explained by the lower adherence of the less

flexible and globular dendrimeric structures to cellular surfaces.

The degree of substitution on the amine functionality has found to

be important as well, with primary amines being more toxic than

secondary or tertiary amines.51,54 This may be explained by the

increased shielding of the positive charge from nitrogen by the

larger molecular size of e.g. alkyl substituents compared to the

hydrogen atoms found in a primary amine.For amino-terminated PPI dendrimers a similar generation

dependent increase in cytotoxicity has been found, with the higher

generation dendrimers being most cytotoxic (IC50 < 5 mg mL21 forG5-PPI).55 As with the PAMAM dendrimers, the PPI dendrimers

showed a generation dependent haemolytic effect on blood cells,

with the high generation dendrimers being most haemolytic.52

In summary, amino-terminated dendrimers are generally cyto-

toxic.52 The cytotoxicity of the cationic dendrimers can be

explained by the favoured interactions between negatively charged

cell membranes and the positively charged dendrimer surface,

enabling these dendrimers to adhere to and damage the cell

membrane, causing cell lysis.

Dendrimers with anionic surface groups: Recent comparative

toxicity studies of anionic and cationic amino-terminated PAMAMdendrimers using Caco-2 cells, similarly conclude that the amino-

terminated PAMAM dendrimers have a significantly higher

cytotoxicity compared to the anionic carboxyl functionalised half-generation PAMAM dendrimers.49 Lower generation PAMAMdendrimers having anionic (e.g. carboxylate) surface groups, show

neither haematotoxicity nor cytotoxicity at concentration of 2 mg

mL21.52 However, the biocompatability of dendrimers is not solely

determined by the nature of their surface groups. Dendrimers based

on an aromatic polyether skeleton having anionic carboxylate

groups on the dendrimer surface have been shown to be haemolytic

on a solution of rat blood cells after 24 h. It is suggested that the

aromatic interior of the dendrimer may cause haemolysis through

hydrophobic membrane contact.52

Surface derivatisation and cytotoxicity: Upon partial derivatisa-tion of the PAMAM dendrimer surface amines with chemically

inert functionalities like PEG or fatty acids the cytotoxicity towards

Caco-2 cells is reduced significantly (from IC50 ~ 0.13 mM to > 1

mM). This can be explained by reduction of the overall positive

Fig. 6 Variation in molecular density of a dendrimer due to back-folding.Increased back-folding leads to an increased molecular density in the coreregion (bottom). The degree of back-folding varies with solvent polarity andpH.

Fig. 7 Surface organisation of a hetero-functionalised dendrimer (top), or ahomo-functionalised dendrimer (bottom), upon interaction with an appro-priate receptor.

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charge when transforming the basic primary surface amino groups

to non-charged amides as well as encapsulating the dendrimer

cationic interior tertiary amines. It was found that partial derivatisa-

tion with lipid or PEG (6 lipid chains and 4 PEG chains on a G4-

PAMAM, respectively) lowered the cytotoxicity. However, upon

introduction of a larger number of lipid- or PEG-chains no

reduction was observed and a high number of lipid chains increased

the cytotoxicity in this system, probably due to cell lysis by

hydrophobic interactions.49

Additives and cytotoxicity: Additives can lead to a significant

reduction in toxicity of amino-terminated dendrimers, where for

example the addition of fetal calf serum together with PAMAM

dendrimers, partially modified with the fluorophore Oregon Green,

reduced the cytotoxicity towards human carcinoma (HeLa) cells in

comparison with the partially modified dendrimer alone.56 In these

systems the toxicity was further reduced by complexation with

oligonucleotides. Studies performed on PAMAM dendrimers

aiming for gene delivery, similarly conclude that DNA complexed

amino-terminated PAMAM dendrimers of low generation (up to

G3) do not possess any significant cytotoxicity in vitro, and that the

toxicity of the dendrimers is reduced upon complexation to

DNA.57,58

Unmodified, amino-terminated PPI dendrimers are similarly lesscytotoxic when formulated with DNA for transfection, with the

lower generation dendrimers (G2) being the best transfection

agents.55 This could indicate that the non-covalent binding between

the dendrimer and protein or DNA lead to a shielding effect of the

polycation, similar to what is obtained by covalent modification of

the dendrimer surface amines.

However, these observations are contradicted by other cytotox-

icity studies on polycationDNA complexes.59 These studies showthe same or higher cytotoxicity when unmodified amino-terminated

G5-PAMAM dendrimer was formulated with DNA; albeit, the

higher toxicity of the DNAdendrimer complexes is not directlyattributed to toxicity of the cationic amino-terminated dendrimer,

but rather to the cellular stress upon transfection with high levels of

DNA (3 mg mL21), which may lead to apoptosis.60 Furthermore,

Gebhart and coworkers suggest that the amino-terminated G5-PAMAM dendrimers still have positively charged amines on the

surface of the dendrimerDNA complex due to their rigidity, whichcould also retain the cytotoxicity of the complex.

2.2.2In vivo toxicity. Only a few systematic studies on the invivo toxicity of dendrimers have been carried out so far. The general

observation is that injections into animals (mice) with 10 mg kg21

concentrations of PAMAM dendrimers (up to G5) do not appear to

be toxic, independent of whether they are unmodified or modified

at the dendrimer surface.16,61 Furthermore, it has been found that

injection of unmodified amino-terminated PAMAM dendrimers

together with ovalbumin in mice did not result in any significant

toxicity in vivo (no weight loss, no granuloma formation, no

haemolysis or inflammation), but that these mixtures had adjuvantactivity.62

Recently, new hydroxy- or methoxy-terminated dendrimers

based on a polyester scaffold (Fig. 8) have shown to be non-toxic

both in vitro and in vivo.63,64 At very high concentrations (40 mg

mL21) these polyester dendrimers induced some inhibition of cell

growth in vitro but no increase in cell death was observed and, upon

Fig. 8 Non-toxic polyester based dendrimer (G4) well-suited for delivery of drugs.63,64

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injection in mice, no acute or long-term toxicity was observed. The

non-toxic properties make these new dendritic motifs very

promising as biodegradable drug delivery devices, as the dendrimer

may be degraded by hydrolytic enzymes after the release of the

drug.

2.2.3 Immunogenicity. Initial systematic studies performed on

unmodified amino-terminated PAMAM dendrimers showed no oronly weak immunogenicity of the G3G7 dendrimers.16,62 How-ever, later studies showed some immunogenicity of these den-

drimers and found that modification of amino-terminated PAMAM

dendrimers with polyethylene glycol (PEG) chains reduces im-

munogenicity and gives longer lifetime in the blood stream in

comparison to unmodified dendrimers.65 The PEG chains increase

the hydrophilicity of the dendrimer, and create a highly hydrated

dendrimer surface with low disturbing effect on the physiological

environment. However, as we shall see, the dendrimer surface can

alternatively be modified with antigens or T-cell helper epitopes

creating highly immunogenic compounds.

2.2.4 Biopermeability. As mentioned, dendrimers complexed

to DNA can be transported into the cell nucleus, with lessmembrane damage and fewer cytotoxic effects compared to free

dendrimer. Several approaches have been investigated to further

increase the transfection ability of the dendrimerDNA adducts,and new dendrimer motifs continue to be developed for that

purpose.58,66In vitro transfection studies concluded that addition of

moderate amounts of sulfonated b-cyclodextrins (b-CDs) en-hances the transfection ability of the PAMAM dendrimerDNAcomplexes, due to ionic binding between the negatively charged

sulfonate moieties of the b-CD and the cationic amino-terminateddendrimer, leading to an altered DNAdendrimer complex compo-sition.67 In polylysine dendrimers, the transfection efficacy was

enhanced by derivatisation of the dendrimer surface with PEG;

however, the overall level of transfection was low, presumably due

to low release of the DNA from the dendrimer.68 The investigationsgenerally conclude that the spherical shape of dendrimers is not an

advantage in gene delivery, which agrees with earlier work, where

fragmented PAMAM dendrimers show superior transfectionefficacy in comparison with the spherical complete dendri-mers.69

In order to use dendrimers for drugs or drug delivery, their

biopermeability on a macroscopic level also has to be taken into

consideration. In vivo studies on the ability of cationic amino-

terminated PAMAM dendrimers (G1G4) to cross the micro-vascular endothelium indicate that the extravasation time across the

microvascular endothelium increases with increasing generation

and molecular weight of the dendrimer.70 Studies on the trans-

epithelial transport of PAMAM dendrimers (G0G4) in Madin

Darby canine kidney cells showed that the G4-PAMAM dendrimerpossessed the largest permeability; however, in these studies no

linear dependence between the dendrimer generation and permea-

bility was found.71 Para-cellular transepithelial transport of amino-

terminated PAMAM dendrimers in a Caco-2 cell monolayer

showed higher permeability for the lower generation dendrimers

(G0G2) in comparison with the higher generation dendrimers,which were also hampered by their increasing cytotoxicity.50 In

vitro studies on anionic PAMAM dendrimers (half-generationPAMAM dendrimers) on an everted rat intestinal sac system

showed that these dendrimers rapidly crossed into the intestine of

adult rats. The transfer rate of the dendrimers was faster than other

polymeric systems, suggesting that these dendrimers could be

useful as building blocks in oral delivery systems.72 Polylysine

dendrimers where the surface has been modified with lipid chainsand studies of their uptake through the intestine of rats concluded

that these lipid modified dendrimers had poorer uptake in

comparison with well-known delivery systems such as polystyrene

latex particles.73

3 Development of hostguest binding motifs in

dendrimers towards biological applications

In the previous section, we considered dendrimer toxicity in

biological environments, and in some cases it was found that

dendrimers can become less toxic upon interaction with additives

such as e.g. DNA; in these cases structurally undefined complexes

between the dendrimer and the DNA are formed. In this section we

will take a closer look at different motifs which are useful forcomplexation between dendrimers and various guest molecules, the

so-called hostguest complexes, and their possible use in drugresearch. The use of dendrimers as hosts or carriers of smaller guest

molecules is a research area of increasing interest and has been

extensively reviewed.7476

3.1 Dendrimers as hosts

PAMAM and PPI dendrimers, with their large molecular size and

multivalent surfaces, serve as good scaffolds for synthetic macro-

molecular hosts, and subsequent surface modification can yield a

host molecule with the desired properties. The hostguest bindingcan either take place in the cavities of the dendrimer core ( endo-receptor), or at the multivalent surface or outer shell of thedendrimer (exo-receptor).74 One early example of an endo-receptor is the dendritic box,7781 where a G5-PPI dendrimer wasmodified at the surface with Boc-protected phenylalanine. In this

way the outer shell was made more dense due to the sterically

demanding Boc-protective groups. Guest molecules of different

size, present during the modification of the dendrimer, were

encapsulated in the interior and isolated from the bulk by the

densely packed Boc-phenylalanine surface. The dendrimer could

simultaneously bind up to 4 large guest molecules (Rose Bengal)

and 810 small guest molecules (p-nitrobenzoic acid). Uponselective acidolysis (formic acid) of the Boc-groups at the surface,

the surface shell became more open and the small guest molecules

were allowed to leak from the dendrimer, whereas the large guestmolecules remained trapped in the core, Fig. 9.

The large guest molecules could subsequently be released from

the dendrimer by acidolysis of the amide bonds creating the

unmodified dendrimer with a more open surface structure. In the

dendritic box, the interactions between the host and the guest

molecule were not tailored to be specific, but more governed by the

molecular size of the guest molecule, and the physical size of the

cavities in the host. By incorporating a biodegradable linkage in the

dendrimer outer shell, the outer shell of the dendrimer host could

alternatively be perforated by physiological or enzymatic hydroly-

sis, directing this host motif towards drug delivery applications.

3.1.1 Guest binding to the dendrimer core via hydro-

phopic interactions. The unimolecular micelle reported byFrchets group is based on a polyaryl ether dendrimeric networkhaving carboxylate surface groups, and is capable of dissolving

apolar guest molecules such as pyrene in water (Fig. 2). The amount

of dendrimer was proportional to the amount of dissolved pyrene.

The hostguest binding is assumed to be mediated through ppinteractions between the electron-rich aryl ether and the aromatic

guest. This was confirmed by the enhanced ability to bind electron-

deficient aromatic guests (p-interaction stabilised) and the decreasein binding of an electron-rich guest molecule (p-interactiondestabilised due to electron repulsion) compared to pyrene.8 These

types of dendrimers would make good candidates for carrying

hydrophobic bioactive compounds, e.g. steroids.

Dendrimers specifically tailored to bind hydrophobic guests to

the core have been created by the Diederich group under the namedendrophanes. The water soluble dendrophanes are centeredaround a cyclophane core, and can bind aromatic compounds,presumably via pp interactions, Fig. 10. These dendritic struc-tures were shown to be excellent carriers of steroids.82,83

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3.1.2 Guest binding to the dendrimer core via polarinteractions. In order to be able to bind more polar bioactivecompounds to the core of a dendrimer, Diederich and coworkers

designed the so-called dendroclefts.84,85 These water-soluble

dendrimers were centered around an optically active 9,9A-spir-

obi[9H-fluorene] core and showed a marked diastereoselectivity

towards recognition of octyl b-D-glucoside over octyl a-D-glucoside, Fig. 11. Proton NMR analysis performed on the host

Fig. 9 The dendritic box.7781

Fig. 10 Left: G3-Dendrophane for the encapsulation of steroids. Right: The hostguest binding motif upon complexation with testostorone.83

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guest complexes showed that hydrogen bonding between the

pyridine carboxamide moieties in the core and the oxygen atoms in

the carbohydrate guest was the major contribution to the hostguestinteractions.

The diastereoselectivity was found to increase with increasing

dendrimer generation, probably due to increased hydrogen bonding

between the bound carbohydrate guest and the alkyl ether oxygen

atoms of the dendritic wedges.84

A simpler approach has been developed by Michell and

coworkers, who modified PAMAM dendrimer surface amines with

a glycerol derivative (tris(hydroxymethyl)aminomethane), thereby

creating water-soluble dendrimers capable of binding acidic

aromatic antibacterial compounds, which could be released by

lowering the pH. The hostguest complex formation occurred viaacidbase interactions and hydrogen bonding between the den-drimer inner tertiary amines and the acidic substrate, however, the

exact nature of the hostguest interactions could not be determinedby 1H-NMR86

3.1.3 Dendrimers as temporary scaffolds. An interestingalternative approach to create hosts with highly defined dendritic

structures has recently been developed by Zimmerman and

coworkers.87 Here the dendrimers can be used as scaffolds for pre-

organised structures in creating artificial hosts by molecularimprinting inside a polymeric network of dendrimers. In thespecific example, a dendrimer consisting of a porphyrin core and asurface containing terminal double bonds was used as monomerand polymerised by Grubbs catalyst into a polydendritic network.Subsequently, the base labile ester bonds between the core and the

dendritic wedges were cleaved, releasing the core porphyrin

structure from the preorganised dendritic polymer (Fig. 12). In this

way, a polymer containing porphyrin-shaped cavities wasobtained and it was shown that this polymer was capable of binding

porphyrins with association constants of 1.4 3 105 M21.87 Thispoly-dendrimer can be regarded as a synthetic porphyrin-recognising antibody.

3.2 Binding to the outer shell of dendrimers the clickin concept

The multi-pincer structure of the PPI dendrimers opens upintriguing possibilities for binding various guest molecules to the

outer-shell pincers of a surface modified dendrimer. One example

is the urea modified PPI dendrimers used for binding of oxo-anions

to the outer shell urea groups via hydrogen bonding, as shown by

Vgtle and coworkers.88 Meijers group used urea- and thiourea-functionalised PPI dendrimers for binding guest molecules contain-

ing a ureaglycine tail unit.38,39 The guest molecules interact withthe dendritic host by multiple urea (guest)(thio) urea (host)hydrogen bonds and ionic interactions between the glycine

carboxylic acid and the dendrimer outer shell tertiary amino groups.

The concept was baptised the click in mechanism, and it has beensuggested that the acidbase reaction between the dendrimer andguest with subsequent Coulomb-attractions pulls the guest into the

dendrimer, whereas the hydrogen bonding keeps the guest bound to

the host. By intake of urea guests the outer shell becomes

increasingly crowded and dense, hence this hostguest motif couldprovide a non-covalent example of a dendritic box (Fig. 13).

As the urea glycine tail is highly similar to the C-terminus of a

peptide, it was investigated whether the dendrimer could act as a

host or carrier for peptides, directing the click in motif towardsbiological applications. This hostguest motif can be useful as a pHsensitive drug delivery system, where the peptide guest can be

released upon lowering pH, as a result of protonation of the

carboxylic acid moiety. It was found that the urea and thiourea

modified dendrimers were capable of binding different peptides,

regardless of the bulkiness of the side chains, and that the peptides

could be released from the dendrimer under mild acidic condi-

tions.89,90 As the dendrimer binds different peptides without

selectivity, this would introduce the possibility of using thedendrimer as host (bus) for several different peptides (pas-sengers) simultaneously, Fig. 14.

4 Dendrimers as drug delivery devices

The research in dendrimer mediated drug delivery has mainly been

focused on the delivery of DNA drugs (genes or gene inhibitors)

into the cell nucleus for gene or anti-sense therapy, and numerous

reports have been published on the possible use of unmodified

amino-terminated PAMAM or PPI dendrimers as non-viral gene

transfer agents, enhancing the transfection of DNA into the cell

nucleus.57,58,9195 The exact structure of these hostguest bindingmotifs has not been determined in detail, but is presumably based

on acidbase interactions between the anionic phosphate moietiesin the DNA backbone and the primary and tertiary amines in thedendrimer, which are positively charged under physiological

conditions. It has been found that partially degraded (or frag-

mented) dendrimers are better suited for gene delivery than the

Fig. 11 G2-Dendrocleft (left), which can act as a diastereoselective host for octyl-b-D-glucoside (right) over the a-anomer.84,85

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Fig. 12 The use of porphyrin core dendrimers as scaffolds in creation of porphyrin binding hosts by molecular imprinting. 87

Fig. 13 The click-in design. X = O,S. Top: Intake of urea containing guestmolecules in urea or thiourea hosts.38,39 Bottom: Intake ofN-Boc-protectedpeptides.89,90

Fig. 14 Investigation of hostguest hydrogen bonding interactions in click-in between a peptide guest and a thiourea modified dendrimer in CDCl3 by1H-NMR (top) and IR (bottom), a) Dendrimerpeptide complex, b)uncomplexed peptide and c) uncomplexed dendrimer. The increasedhydrogen bonding upon complex formation results in a downfield shift inNMR and a shift towards lower wavenumbers in IR.89,90

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complete dendrimers (vide supra), and a fragmentation (activation)

step consisting of hydrolytic cleavage of the amide bonds is needed

to enhance the transfection efficiency, Fig. 15.69,96,97

In comparison to the intact dendrimers, the partially degraded

dendrimers have a more flexible structure (fewer amide bonds) and

form a more compact complex with DNA, which is preferable for

gene delivery by the endocytotic pathway.97 In addition, it is

generally found that the maximum transfection efficiency is

obtained with an excess of primary amines to DNA phosphates,yielding a positive net charge of the complexes. The more flexible

higher generation PPI dendrimers (containing no amides) are found

to be too cytotoxic for use as non-viral gene vectors, however, the

lower generations are well-suited for gene delivery.55

The unmodified amino-terminated dendrimers transport the

DNA to the cell membrane and may help in the transfection process

by disruption of the cell membrane. The transfection of free DNA

will be hampered by electrostatic repulsion between the negatively

charged phosphate groups in the DNA backbone and the negatively

charged cellular membrane, Fig. 16.

Cationic, amino-terminated dendrimers which are partially

covalently modified with drugs, can be useful as extracellular

stickers in extracellular matrix-targeted local drug delivery,giving a very high local concentration of the carried substrate ordrug close to the cellular surface.98 However, this drug (gene)

delivery technique is only appropriate if a particular drug or gene

has to be introduced into a broad range of cells. In order to obtain

a specific cellular treatment, drug vehicles that direct the drug only

to specific cell types can be designed. One example of such cell-

specific dendritic drug vehicles is a dendrimer derivatised with folic

acid (pteroyl-L-glutamic acid). Folic acid is an important substrate

for uptake in cells by the folate receptor pathway. As the folate

receptor is over-expressed in cancer cells, these folic acid

derivatised dendrimers are taken up by cancer cells preferentially to

normal cells, making these dendrimers well-suited for the cancer-

specific drug delivery of cytotoxic substances.99,100 Very recently,

folate modified PAMAM dendrimers have been successfully used

as carriers of boron isotopes (10B) in boron neutron-capturetreatment of cancer tumors.101 An earlier report concluded that the

use of unmodified amino-terminated PAMAM (Starburst) den-drimers as boron carriers in combination with monoclonal

antibodies had low in vivo tumor localising properties and gave rise

to accumulation in the liver.102,103PAMAM dendrimers conjugated

to the well-known anticancer drug cis-platin act as macromolecular

carriers for platinum. The dendrimer-platinate gives a slower

release of the platin, and shows higher accumulation in solid tumors

and lower toxicity compared to cis-platin.104 PAMAM dendrimer

silver complexes that slowly release silver have shown anti-

microbial activity against various Gram positive bacteria.105The above mentioned approaches to drug delivery are based on

non-covalent complex formation between the drug and the

dendrimeric drug carrier. Another approach is to bind the drug

covalently to the multivalent dendritic surface, via a biodegradable

bond. Dendrimers based on a 1,4,7,10-tetraazacyclododecane core

having primary amines at its surface have been partially modified

with 1-bromoacetyl-5-fluorouracil to form a labile imide linkage.

Upon hydrolysis of the imide under physiological conditions, the

potent antitumor agent 5-fluoro-uracil could be released in

vitro.106

4.1 Dendrimers as glycocarriers

Carbohydrates constitute an important class of biological recogni-

tion molecules. They differ from polypeptide recognition mole-cules in displaying a wider variety of spatial structures (due to

possibilities of branching and anomericity) with fewer building

blocks resulting in a high specificity combined with quite low

binding affinity constants, often in the 10251026 M range.107Carbohydrate binding proteins are called lectins108 and lectincarbohydrate interactions have been described in numerous cases in

the immune system (cellular activation events), in bacterial and

viral infections, in relation to cancer and cell growth etc.

Carbohydrate-based drugs are therefore of interest as microbial

Fig. 15 Activation of a G5-PAMAM (Starburst) dendrimer by solvolysis, leading to partial hydrolysis of amides in the dendrimer.

Fig. 16 Proposed scheme for the transfection of DNA into the cell nucleusaided by activated dendrimers.

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anti-adhesins, microbial toxin antagonists, anti-inflammation

drugs, antiviral and anticancer drugs. However, there are synthetic

difficulties in obtaining such bioactive carbohydrate ligands. A

strategy to overcome this utilises the multivalency/cluster effect

(vide supra) obtainable by dendrimer presentation, to create

carbohydrate ligands with adequate binding affinities from simple

mono- or oligosaccharides. In biologically relevant structures,

multivalent interactions between relatively short saccharides and

lectins/carbohydrate receptors, (which as a rule contain multiple,identical carbohydrate binding sites) have also been observed and

made possible by multivalent presentation of saccharide binding

determinants, thus taking advantage of the cluster effect. This is

seen for example in glycoproteins with multiple, identical anten-nae emanating as branches from a central attachment point. Someof these so-called glycans in fact resemble dendrimers verymuch, even down to the geometrical increase in terminal saccharide

units from G0 to G3, Fig. 17107. Multivalency may also beachieved by lateral rearrangement of individual glycoconjugates,

e.g. glycolipids in cell membranes. An estimate of the distances

between the terminal monosaccharide units in such glycans has

been attempted109 for oligomannoside triantennary glycopeptide

structures which mimic mammalian high-mannose glycans. The

maximum distance attainable between the oligosaccharidebranches was found to be below 30 (3 nm).

A recent review of carbohydrate receptors in nature with an

emphasis on multivalency and the importance of the cluster effect

is found in a review by Bezouska,110 and several reviews

concerning the biomedical use, design and synthesis of glycoden-

drimers with a thorough review of a number of neoglycoconjugate

types have been published.9,111113

Generally, low-valency neoglycoconjugates (up to 16 saccharide

units) give highly enhanced affinities while high-valency neoglyco-

conjugates, although having enhanced binding affinities, may also

lead to unwanted immunological reactions. Low-valency con-

structs are inherently easier to analyse and errors will more easily

appear in high-valency constructs. For example, MALDI-TOF MS

and NMR data on mannose-functionalised PAMAM dendrimers(G2G7) shows that incorporation efficiency decreases withincreasing generation (G5G7 give non-stoichiometric incorpora-tions) which is attributed to dendrimer defects rather than problems

with non-quantitative incorporation yields. NMR does not identify

these problems, probably because of the high degree of symmetry

in the glycodendrimer.114

Especially useful, non-immunogenic glycoconjugates include

peptide supported saccharides and dendrimer-supported sacchar-

ides (glycodendrimers).111 Such adducts have been prepared from

G3-PAMAM or multibranched lysine dendrimers, e.g. by coupling

between mannose isothiocyanate (or sialic acid, lactose and 3A-sulfo

Lewisx saccharides) and the terminal amines of the dendrimer.

Enhancement of affinity towards a macrophage/monocyte localised

mannose-specific lectin was 300400 times for the octamericadduct, compared to the monosaccharide (mannose).

Other very efficient glycoconjugate receptor binders are trivalent

peptideN-acetyl galactosamine and galactose adducts, which bindwith very high affinity to the hepatic asialoglycoprotein receptor.

These glycoconjugates present three identical monosaccharides

bound through spacers to a peptidic backbone at evenly spaced

attachment points.115 The asialoglycoprotein receptor is located in

the cell membrane of hepatocytes and is an example of a multimeric

mammalian lectin.116 This receptor was shown previously to be

specific for galactose and galactosamine and has a clear preference

for desialylated, multiantennary oligosaccharides of many serum

glycoproteins, binding with the following preference: triantennary

> biantennary > monoantennary.117,118 This represents a natural

example of the cluster effect, showing several-thousand fold

affinity increase going from one to three-ligand clusters. As the

asialoglycoprotein receptor is important for adherence to-, andtranfection through- the cell membrane, new transfection agents

based on combinations of nucleotides and glycoside clusters have

been designed to bind and eventually to be internalised by this

receptor (vide supra).

Other types of glycosylated dendrimers, their synthesis and their

applications as antigens for diagnostic, vaccine and biochemical

have been extensively been reviewed elsewhere119,120

Using PAMAM dendrimers (G2G7)114 the different genera-tions of mannose terminated dendrimers were compared with

respect to relative binding activities towards the mannose-binding

plant lectin concanavalin A (con A) in a hemagglutination

inhibition assay. Con A has mannose-binding sites spaced by 6.8

nm on both sides of the tetrameric protein. It was found that 8- and

16-mer glycodendrimers did not show any increase in activity (permonosaccharide entity) compared to the monosaccharide (me-

thylmannoside), which was not surprising as these small den-

drimers do not span the two binding sites of con A and thus no

clustering effect could be expected. Surprisingly, the 32-mer

mannose PAMAM (G4) had a higher relative activity, indicating a

clustering effect, even though theoretically, the carbohydrate units

are not spaced far enough to ensure multivalency towards the

receptor. With the higher generation dendrimers multivalent

binding was clearly indicated, although in the higher generations,

steric effects might play a role in decreasing the interaction

strength.

Glycodendrimers with the cancer-associated T-antigen disaccha-

ride (bGal 13 aGalNac) present in 26 copies were synthesized

based on N,NA-bis(acrylamido)acetic acid cores, and tested forbinding to the galactose-specific lectin peanut agglutinin and a

mouse monoclonal antibody directed against the T-antigen, and a

valency dependent increase in affinity from 106 M21 (monomer) to

108 M21 (tetramer) was demonstrated. Interestingly, the hexamer

showed a substantially lower overall apparent affinity, indicating

that steric effects participate in addition to the valency effect.121

This type of construct has potential application in the detection of

malignant tissues expressing T-antigen receptors, and, ultimately

for targeting drugs to such altered tissue (one example being breast

cancer carcinomas). Moreover, these glycodendrimers are non-

immunogenic and show a superior level of molecular definition

(monodispersity) compared to other types of glycopolymers.

Similar constructs with similar properties but based on PAMAM

(G1G4) have also been reported.122 The synthesis and use inbreast cancer therapy of such T-antigen containing dendrimers has

been recently reviewed.123

Mannosylphenyl-functionalised PAMAM dendrimers (G1G4)and their interactions with lectins (con A and PSA), were compared

to those of monosaccharides in an inhibition assay.124 The

mannosylated dendrimers were up to 400 times more inihibitory

than methyl-mannose monosaccharide, indicating a strongly dendr-

Fig. 17 Dendrimeric N-bound glycan (4-branched antenna).107

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itic effect (a G4-PAMAM dendrimer carries 32 mannose surface

units). However, no big increase in affinity was observed from the

8-mer (G2) to the 16 (G3) and 32-mers; most of the increase took

place going from monomer to dimer and to the tetramer and 8-mer.

The 8-mer generally showed the highest affinity increase per

mannose residue (28 times increase for both lectins). In a very

similar study, but emphasizing the significance of the precise

spatial arrangement of identical disaccharide moieties on the

binding to different types of sugar-binding biomolecules, Andrand coworkers125 concluded that multivalent presentation of the

carbohydrate units clearly increased the binding affinity towards

plant lectins, while a lactoside-specific antibody bound less

efficiently. Binding was also compared to binding of a conventional

neoglycoprotein molecule containing the same carbohydrate con-

stituents but having a more heterogenous presentation of the

carbohydrate moieties. This was bound most efficiently by the

antibody. These types of arylic PAMAM glycocarriers124,125

adhere readily to plastic and are suitable for use as antigens in

common solid-phase assays (e.g. ELISA).

In a structurally quite complicated version of the glycocarrier

dendrimer theme Baussanne and coworkers126 describe the synthe-

sis of a decorated b-cyclodextrin, the b-cyclodextrin being

conjugated in one position (at a primary hydroxyl group) with adendritic branch (a dendritic wedge) terminating in mannoseresidues; hereby a highly asymmetrical dendrimer is obtained

which combines the hydrophobic drug carrier ability of b-cyclodextrin with the multivalent display of a biologically

interesting monosaccharide. The use of a dendritic branch allowsthe attachment of multiple carbohydrate ligands to a single

cyclodextrin molecule, without destroying the integrity and

physicochemical properties of the cyclodextrin molecule, thus still

allowing it to act as a hydrophobic drug carrier in aqueous solution.

In this kind of construct, the dendritic branch can be said to function

as a spacer separating a ligand cluster from a bioactive constituent

(drug-carrier complex).

Reaction with the mannosyl-specific lectin concanavalin A was

analysed by an inhibition solid- phase assay and yielded dissocia-tion constants in the 10241026 M range, increasing with themultimericity of the construct (from mono- to hexavalent), while

the cyclodextrin moiety retained its ability to solubilise a

hydrophobic drug. A fine analysability (NMR and mass spectrome-

try) was shown by 3,5-di(2-aminoethoxy)benzoic acid based

dendrimers (G1G3) decorated with lactose via a thiourealinkage.127 Furthermore, there was a substantial increase in affinity

for the lactose-specific (GM1 ganglioside specific) cholera toxin B-

subunit (500 times increase for the octamer compared to monomer)

in a soluble fluorescence assay. This might constitute an interesting

route for the preparation of cholera therapeutics. These authors

show, however, that the contribution to the affinity increase from

the thiourea and phenyl groups is more than 70 times, almost by

itself accounting for the total synergetic effect of the tetramer andhalf the synergetic effect of the octamer. In another study onbacterial carbohydrate-binding toxins (cholera toxin and Echer-

ichia coli heat-labile toxin), the direct design of a pentavalent

inhibitor glycodendrimer was described, based on the pentavalent

binding of these types of toxins to cell surface glycolipids (GM1

gangliosides), and retaining the geometry of the natural ligands.128

Also in this study the surface monosaccharides are combined with

an aromatic substituent leading to an increased affinity. A high-

affinity, non-aggregating interaction with the toxins was achieved

and was characterised by X-ray diffraction and shown to be very

similar to the structure of the toxin with its natural ligand. The

scaffold used here was not a traditional dendrimer, but rather a

coupling to a pentameric core-linker molecule.128 Again, the

importance of including an aromatic substituent in the ligand wasshown as well as the exact geometry of the construct, especially the

length of the arms, and this could bring the affinity constant of theinhibitor into the range of the natural ligand which is veryimportant if such an inhibitor were to have any therapeutic use. The

use of this type of toxin inhibitors is of some interest as there is no

demand for them to cross the intestinal barrier and to enter the blood

stream, as they are to block the interaction of the toxin with

epithelial cells within the intestines.128

G1G4 PAMAMs were used for the attachment of a modifieddisaccharide which was transformed into an active ester and then

coupled to the surface amino groups, the disaccharide being the

clinically important T-antigen (Gal b13 GalNAc), which ischaracteristic of certain cancers, in particular breast cancercarcinomas.129 The idea was to use such constructs for interaction

with carcinoma-related T-antigen binding receptors (thereby inter-

fering with carcinoma growth) or to use them for generation of T-

specific antibodies for diagnostic and therapeutic purposes. In an

enzyme-linked immunoassay (ELISA) it was shown that the G4-

adduct bound twice as much monoclonal antibody as the G3-adduct

and four times as much as the G2-adduct. Interestingly, the G2-

adduct (8-mer) was more than 25 times as efficient as the G1-

adduct (tetramer), indicating a real dendritic effect going from G1

to the G2 PAMAM dendrimer, while no further dendritic

enhancement was seen with G3 and G4 dendrimers. In a

competition ELISA, 50% inhibition demanded approximately 500

times more monomer than G1-adduct (molar comparisons); on a

per saccharide basis the inhibitory efficiencies of the dendriticstructures were all similar and approx. 100 times the monomer. The

discrepancy between the indirect and the blocking ELISAs goes

uncommented (the tetramer being comparatively much less active

in the indirect ELISA) but could be due to differences in coating

abilities of the different dendrimer constructs which is also not

discussed.

In conclusion, numerous studies have shown that glycosylated

dendrimers are good mimics of natural glycoconjugates and will

interact efficiently with natural carbohydrate receptors, in many

cases to an extent that allows competition with natural binding

substances.

5 Dendrimer drugs

5.1 Dendrimers as antiviral drugs

In general, antiviral dendrimers work as artificial mimics of the

anionic cell surfaces, thus the dendrimers are generally designed

having anionic surface groups such as sulfonate residues or sialic

acid residues, which are acidic carbohydrates present at the

mammalian cell surface. In other words, the dendritic drug

competes with the cellular surface for binding of virus, leading to a

lower cell-virus infection probability, see Fig. 18.

Polylysine dendrimers modified with naphtyl residues and

having sulfonate surface groups have been found to be useful as

viral inhibitors for Herpes Simplex virus in vitro.61 The dendrimer

works both as an inhibitor for virus entry and in late stages of virusreplication.130 PAMAM dendrimers covalently modified with

naphthyl sulfonate residues at the surface, giving an polyanionic

surface, also show antiviral activity against HIV. Also here the

dendrimer drug works as an inhibitor for early stage virus/cell

adsorption and at later stages of viral replication by interfering with

the reverse transcriptase and/or integrase enzymes.131,132 PAMAM

dendrimers derivatised with sialic acid are efficient inhibitors of

infection with influenza A subtype H3N2, however the inhibition is

restricted to this influenza subtype.133 A dendrimer with an amide

surface has been designed and works as an inhibitor for the

respiratory syncytial virus (RSV), see Fig. 19. The exact mecha-

nism of action has not been revealed in detail, but may rely on

hydrogen bonding interactions between the viral fusion protein and

the dendrimer surface groups, causing inhibition of virus bindingand fusion. However, even small alterations at the aromatic

residues of the dendrimer decrease the antiviral activity and viral

selectivity, suggesting that other binding modes e.g. pp stackingcould play a role as well.134

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5.2 Dendrimers as antibacterial drugs

In contrast to the antiviral dendrimers, the antibacterial dendrimers

generally contain cationic surface functionalities such as amines or

tetraalkyl ammonium groups (Fig. 19). The general mode of action

of the antibacterial dendrimer is to adhere to and damage the

anionic bacterial membrane, causing bacterial lysis. PPI den-

drimers where the surface has been functionalised with tertiary

alkyl ammonium groups have shown to be very potent antibacterial

biocides against Gram positive and Gram negative bacteria.135137

The nature of the counter ion is important, as tetraalkylammonium

bromides were found to be more potent antibacterials over the

corresponding chlorides.136 The dendritic biocides were found to

have higher activity in comparison with other hyperbranchedpolymers. Polylysine dendrimers having mannosyl surface groups

have been shown to inhibit adhesion ofE. coli to horse blood cells

in a haemagglutination assay, making these structures promising as

antibacterial agents.138

5.3 Dendrimers as antitumor drugs

In photodynamic treatment (PDT) the drug becomes toxic upon

irradiation by in situ formation of small amounts of singlet oxygen,

which has strong physiologically damaging effects.139 In compar-

ison, the drug should be relatively non-toxic under non-irradiative

conditions (low dark toxicity), thus acting as a prodrug under non-irradiative conditions, see Fig. 20. Few reports have been published

so far on the design of dendrimers containing various photo-

sensitizers for the formation of singlet oxygen in the tumor tissue,but this research area may grow rapidly over the coming years.

Dendrimers containing the photosensitizer 5-aminolevulinic acid at

their periphery have been synthesised and are promising as agents

for PDT of tumorigenic keratinocytes.140 Polyaryl ether based

dendrimers derivatised with the photosensitizer protoporphyrin,

have been evaluated as candidates for the PDT of solid tumors. 141

The protoporphyrin derivatised dendrimers showed more specific

cytotoxicity than protoporphyrin itself, and the dendrimers were

more potent upon irradiation compared to protoporphyrin, probably

due to an antenna effect of the dendritic wedges. The dendrimers

showed a 140-fold lower dark-toxicity, compared to free proto-

porhyrin, a low dark toxicity is an important requirement in PDT,

as high dark toxicity causes unspecific cytotoxicity.

6 Dendrimers as protein denaturants

Certain types of dendrimers act as chaotropes i.e. water structure

perturbing solutes, lowering the dielectric constant and the

Fig. 18 Cartoon showing the anti-viral action of dendrimer drugs. Binding of the virus to the cell membrane, and subsequent penetration of the cell wall by

the virus (endocytosis) is inhibited by the sialic acid decorated dendrimer drug.

Fig. 19 Examples of molecular structures of anti-viral or anti-bacterialdendrimers. Top: Antiviral dendrimer, Respiratory Syncytial Virus.134

Middle: Antiviral dendrimer, Herpes Simplex Virus.61 Bottom: Anti-bacterial dendrimer,E. coli.136

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viscosity of water; disordering the regular water structure by

reorganising water molecules at the dendrimer surface. As with

other chaotropes, this leads to hydrophobic interactions being

disfavoured which, in turn, is highly destabilising for most protein

tertiary structures (denaturation). Classical examples of chao-

trophic salts are MgCl2, urea, guanidinium chloride, sodium

thiocyanate, guanidinium thiocyanate at high concentrations and

other chaotropes include polarity-decreasing, water miscible or-

ganic solvents such as acetonitrile, propanol and methanol.

Generally, chaotropes will serve to denature and solubilise proteins,

which is useful for example in solubilising protein aggregates as are

often encountered when expressing proteins in heterologousexpression systems (inclusion body formation) and when extracting

certain types of membrane proteins. Dendrimers, being compact,

large polyionic substances have the physicochemical properties

needed to make them potential chaotropes/protein denaturants,

A striking example of this was reported recently in the

literature,142 where cationic dendrimers were used for the sol-

ubilisation of prion protein aggregates. Prion proteins are able to

attain a pathogenic structure/conformation in which they can cause

mortal diseases called spongiform encephalopathies, including mad

cow disease and Creutzfeldt-Jakobs disease. These deadly con-formers are characterised by their tendency to form very insoluble

aggregates, which are found in the brains of affected individuals.

Such aggregates are soluble only in solvents containing bothdetergent and denaturant (typically 6 M guanidinium chloride);

however it was shown that such aggregates can be solubilised by

cationic dendrimers, such as PEI-, PPI- and PAMAM dendrimers,

higher generation ( > G3) dendrimers being the most efficient and

influenced by the number of surface amino groups. PAMAM

dendrimers having hydroxy groups at their surface (PAMAM-OH)

and linear polymers had no or very minor effects. The effect was

seen at surprisingly low concentrations (7 mg ml21 or below) onaggregate producing neuroblastoma cells and took place with no

cytotoxicity.7 Other types of compounds capable of dissolving

already formed aggregates of the prion protein have not been

described.

7 Dendrimers in vaccines

It is well-established that small molecular weight substances (e.g.

peptides) are not very immunogenic. i.e. no or a weak immune

response (including antibody formation) is induced upon their

injection into a recipient host. However, this problem can be

overcome by increasing the molecular weight of the substance in

question either by polymerisation or by coupling it to a multi-

functional, high molecular weight carrier, traditionally a naturally-

derived protein.19 For the preparation of highly defined, reproduci-

ble immunogens, e.g. for human vaccine uses, other types of

carriers are highly desirable and in this respect, dendrimers have

emerged as useful since they can act as multivalent and well-

defined carriers for antigenic substances by coupling of antigen

molecules to the surface functional groups of the dendrimer.Examples of dendrimerpeptide compounds being used for

vaccine and immunization purposes include the multiple antigenic

peptide (MAP) dendrimer system pioneered by Tam and cowork-

ers,14,143,144 which can be synthesised with defined mixtures of B-

and T-cell epitopes,145 either synthesized by stepwise peptide

synthesis on the branches of the MAP or by segment coupling of

peptide fragments by various methods.143 By far, most of the

reported immunizations with MAP constructs have been performed

with traditional adjuvants as e.g. in the work by Moreno and

coworkers146 in which aluminium hydroxide, Freunds adjuvant,and a saponin adjuvant (QS-21) were tested for the ability to induce

antibodies together with a MAP structure containing Plasmodium

falciparum T- and B-cell stimulatory peptides. Using cancer related

peptides, Ota and coworkers showed that MAPs were processed inantigen-presenting cells in the same way as antigens derived from

intracellular pathogens (e.g. viruses), thereby providing a powerful

immune response, including cytotoxic T-cells.147

The MAP construct is a wedgelike, asymmetrical dendrimer type

formed by building successive generations of lysine residues

acylating the a- and e-amino groups of the preceding lysineresidues. This results in a structure displaying an equal number of

non-equivalent a- and e-primary amino groups that can be coupledto a small molecular weight antigen of interest, with the purpose of

rendering the antigen immunogenic and obviating the need to use

carrier proteins. The most preferred MAP-structures for vaccina-

tion purposes are tetra- or octameric. By using orthogonal

protection strategies, different types of antigenic moieties may be

coupled in a controlled fashion to the same MAP carrier, Fig. 21.A simple peptide carrier is based on a tetrameric MAP dendrimer

in which cyclised antigenic peptides have been coupled and in

which lipidic moieties are present in the core. Although no

immunological data have yet been presented it is stated that this

Fig. 20 Schematic depiction of photo dynamic therapy (PDT) using adendrimer with a protoporphyrin photosensitizer core, which uponirradiation with light and subsequent reaction with oxygen creates tissue

damaging singlet oxygen.

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construct was a very promising foot and mouth disease vaccine

candidate.148

MAP structures have been used in a large number of studies for

producing peptide-specific antibodies143 and are also being devel-

oped for vaccine use, a MAP-based malaria vaccine being now in

phase I human trials.146,149,150

The MAP carrier has also been used for the preparation of non-peptide (carbohydrate, haptens) antigens for vaccination purposes.

An example of the MAP system for glycoimmunogens is the Tn-

antigenic dendrimer studied by Bay and coworkers151 where a

tetrameric core structure is derivatised with the Tn-antigen and with

a Th-cell stimulatory peptide and was shown to react with

monoclonal antibodies against Tn, Fig. 22. A different version of

this, containing trimeric Tn-building blocks was later shown to be

immunogenic,152 and useful for active immunization against colon

carcinomas in BALB/c mice, using alum as the adjuvant. As the

mono-Tn analogue was less efficient than the tri-Tn analogue and

as a linear analogue containing two tri-Tn moieties was also less

efficient, it was concluded that the precise spatial arrangement and

clustering of the Tn-epitope was very important for the im-

munogenicity. G5-PAMAM (Starburst) dendrimers have beenapplied as carriers of the Tn-antigen and the resulting glycoconju-gates were tested as vaccine candidates in comparison with a carrier

protein (ovine serum albumin) conjugated to a monomer, dimer or

trimer of the Tn-antigen. It was found that the Tn-antigendendrimer conjugates elicited no antibody response, and hence no

immunogenicity, whereas Tn-antigen conjugated with a carrier

protein or lipopeptide gave rise to antibody responses. The Tn-

dimer lipopeptide conjugate also gave rise to IgG antibodies.153

Another peptide carrier system which is not dendrimeric per se

but becomes a dendrimeric structure upon derivatisation with

peptides is the nondendritic peptide carrier by Heegaard and

coworkers,154

in which the attachment points for the peptidebranches are designed to space the attached peptides in an optimal

fashion and to allow the peptide backbone to attain some structure

in aqueous buffer, lending a certain degree of conformational

definition to the whole complex and thereby supporting structural

trends in the attached peptides. This phenomenon of organisation-

ally induced structure has previously been demonstrated by

Tuchscherer and coworkers155 in the so-called template assisted

synthetic peptides in which four identical peptides are coupled to a

tetrafunctional, cyclic template, leading to an increased conforma-

tional definition of the peptides, compared to the peptides alone.

This peptide construct was shown to be immunogenic in the

absence of adjuvant and furthermore, in contrast to MAP, had high

aqueous solubility.154

McGeary and coworkers have prepared carbohydrate-based(glycolipid) dendrimers as potential carriers for peptide antigens,

utilising the multihydroxy functionalities of a single mono-

saccharide as the basis for multimeric presentation of antigens and

showing the applicability of solid phase synthesis for this

purpose.156 Although no actual peptidedendrimer constructs were

Fig. 21 Schematic depiction of different MAP-designs comprising different peptides representing T- and B-cell epitopes, respectively and showing differentways to organise these elements in MAP structures. Also shown is the possibility of including fatty acids into such structures either as single, straight chainalkyls or as the specific tripalmitatecysteinyl structure (N-a-palmitoyl [2,3-bis(palmitoyloxy)propyl]cysteine.

Fig. 22 Structure of a Tn-antigen based MAP immunogen comprising peptides representing T-cell stimulating epitopes in addition to trimeric Tn-epitopes(Galactose-threonine),151 the T-cell epitope peptide being Lys-Leu-Phe-Ala-Val-Trp-Lys-Ile-Thr-Tyr-Lys-Asp-Thr (from the poliovirus VP1 protein).

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synthesized and no immunization experiments were done, the use

of carbohydrate functionalities for multimeric presentation of

antigens is clearly warranted, and the possibility of including such

structures into glycodendrimers (vide supra) for immunogen

construction is presented.

Baek and coworkers explicitly claim that multimeric carbohy-

drate moieties as presented in glycodendrimers are non-im-

munogenic.129 These and other examples indicate that multi-

mericity is not enough by itself to render carbohydratesimmunogenic, somewhat in contrast to peptide antigens (vide

supra).

The use of dendrimers as adjuvants has been described by

Rajananthanan and coworkers.62 Here, the ability of different

aggregate formulations to act as adjuvants is studied. Adjuvants are

substances which augment the immune response to an antigen when

both are administered together, and such compounds are virtually

indispensable for the manufacture of efficient vaccines. Rajanan-

thanan compares two glycolipid-containing aggregates with a G5-

PAMAM dendrimer (5 nm diameter). The idea is, that amphilicity

is related to protein carrying potential and thereby adjuvanticity. As

such, the glycolipids of this study were to be expected to be much

more amphiphilic than the dendrimer and moreover, they were

meticulously formulated with various other components to preparemultimolecular complexes with non-covalently entrapped antigen

ad modum Iscoms.157 However, when testing immunogenicity inmice with a standard protein antigen (ovalbumin), mixing antigen

and dendrimer increased the immune response above that seen

when administering the antigen alone, reaching titres in the 105

range being 10 times the titres reached with the antigen alone.

Wright claims that G3-PAMAM and other mid-generation

dendrimers can be used as adjuvants for vaccine purposes when

used in a dilution which ensures the absence of toxicity.158

Conclusion

Dendrimers are extremely well-defined, globular, synthetic poly-mers with a number of characteristics which make these polymers

useful in biological systems. Dendrimers have unique properties

enabling them to respond to changes in solvent conditions in a

predictable manner and can easily be modified to act as highly

specific binders of various biological substances. Dendrimers can

be tailored or modified into biocompatible compounds with low

cytotoxicity and high biopermeability etc. In addition, dendrimers

are manufactured in high purities with few structural defects, and

are easily analysed by standard methods as mass spectrometry,

infrared spectroscopy and NMR spectroscopy.

As described, a number of dendrimer types are now commer-

cially available and have already found use as drug candidates for

receptorligand interactions, drug carriers for conferring bio-

survival, membrane permeability and targeting, and have foundwide use as carriers for vaccine antigens as well. Furthermore,

dendrimers have proven very useful as scaffolds in the design of

biosensors, imprinting scaffolds and artificial receptors (specific

hostguest interactions).It is to be expected that new dendrimer structures will continue

to be developed, and more types of dendrimers will be manu-

factured to a high degrees of perfection and will become

commercially available. Finally, new methods and strategies for

synthesising, modifying and derivatising dendrimers will be

developed, enabling specific tailoring of binding motifs, charge

density etc.

This will add a new degree of sophistication to well-known drugs

and reagents, as well as creating entirely new classes of drugs and

bioactive substances based on these macromolecular, yet beauti-fully simple, structures.

Acknowledgements

The authors would like to thank Dr. Edward Farrington for proof

reading this manuscript and Johan Fleischer for assistance in

creation of front cover graphics. The Danish Agricultural and

Veterinary Research Council (Grant no. 23-02-0011) are acknowl-

edged for financial support.

References

1 K. Autumn, Y. A. Liang, S. T. Hsieh, W. Zesch, W. P. Chan, T. W.Kenny, R. Fearing and R. J. Full,Nature, 2000, 405, 681685.

2 E. Buhleier, W. Wehner and F. Vgtle, Synthesis, 1978, 155158.3 R. G. Denkewalter, J. F. Kolc and W. J. Lukasavage; Patent

US4410688, 1983.4 D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J.

Roeck, J. Ryder and P. Smith, Polym. J., 1985, 17, 117.5 E. M. M. de Brabrander van den Berg and E. W. Meijer, Angew.

Chem., Int. Ed. Engl., 1993, 32, 13081311.6 G. M. Dykes,J. Chem. Technol. Biotechnol., 2001, 76, 903918.7 S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner and M.

Scott, Proc. Natl. Acad. Sci., USA, 1999, 96, 1452914534.8 C. J. Hawker, K. L. Wooley and J. M. J. Frechet,J

Documents

Ulrik Boas and Peter M. H. Heegaard- Dendrimers in drug research